cis–trans Engineering: Advances and Perspectives on Customized Transcriptional Regulation in Plants  Ankita Shrestha, Ahamed Khan, Nrisingha Dey  Molecular Plant  Volume 11, Issue 7, Pages 886-898 (July 2018) DOI: 10.1016/j.molp.2018.05.008 Copyright © 2018 The Author Terms and Conditions

Figure 1 Approaches to cis Engineering Depicted by an Engineering Analogy. Environmental stimuli trigger primary signals (indicated as dotted arrows), which direct the binding of stress-specific TFs upon their respective cis elements (CEs). This CE–TF complex deflects these signals (indicated as solid arrows) to the core promoter, which is the focal point of all impulses within the network. The strength, magnitude, and type of signal recorded by the pre-initiation complex (at the core promoter) in turn regulates the net gene expression. The gene expression patterns are represented as “bars” where poor signal reception (fewer bars) normally indicates lower levels of gene expression while more bars indicate stronger gene expression. The core promoter also interacts with the proximal and distal regions of the promoter by transmitting several intermittent signals. These interactions play a major role in deciding the fate of each motif present in the closer and distant proximities and determine which changes can be manifested within them. Such changes include deletion, addition, substitution, and duplication. Alterations within the proximal region of a promoter change the overall stoichiometry of the motifs present on the DNA sequence. This can be attributed to the changes made in the spacing and copy number of individual CE present in the upstream activation sequence of the promoter, which in turn modulate the gene expression patterns. Molecular Plant 2018 11, 886-898DOI: (10.1016/j.molp.2018.05.008) Copyright © 2018 The Author Terms and Conditions

Figure 2 trans Engineering Using Zinc Fingers and Transcription Activator-like Effector Nucleases. (A) Zinc finger (ZF) variants can be logistically engineered by tethering an activator or repressor domain, thereby providing potential avenues for gene activation (solid arrow) or suppression (dotted arrow), respectively. (B) Engineered transcription activator-like effector nucleases (TALEs) can also be designed by linking them to an activator or repressor domain. The binding domain effector (TALE) protein recognizes its corresponding nucleotide and is more specific than the ZFs in terms of tolerance to mismatches. The activators VP16 or VP16 X4 and repressors such as SRDX, KRAB, or SID domains are commonly used for transcriptional modifications within the cellular system. Molecular Plant 2018 11, 886-898DOI: (10.1016/j.molp.2018.05.008) Copyright © 2018 The Author Terms and Conditions

Figure 3 CRISPR-Cas9-Mediated trans Engineering for Modulation of Endogenous Gene Expression. The CRISPR-Cas9 effector systems have been devised in such a manner that they can be used for either transcriptional activation or repression. This approach employs a catalytically inactive or dead Cas9 nuclease, which is directed to the target site along with an anchored activator or repressor domain. The specificity of this ensemble is exceptionally compelling due to the PAM-directed sequence-specific DNA binding. (A) This system works to silence the target gene expression when the dCas9 sits over the core promoter region (guided by the small guide RNA [sgRNA]), hence blocking the binding of RNA polymerase or other proteins/factors involved in the basal transcriptional machinery. (B) Cas9 is inactivated by either mutating or removing the RuvC or HNH nuclease domain to create dCas9 and fusing it to transcription activators or repressors. A repressor domain such as KRAB-fused dCas9 allows transcription to be further repressed by the process of heterochromatinization. (C) Steric hindrance created by dCas9 halts transcriptional elongation. This is achieved by designing sgRNAs complementary to the template/non-template strand, which directs dCas9 to the specific promoter region and restricts the movement of RNA polymerase. Molecular Plant 2018 11, 886-898DOI: (10.1016/j.molp.2018.05.008) Copyright © 2018 The Author Terms and Conditions

Figure 4 Engineering dCas9 for Epigenetic Modifications. dCas9 can be coupled to epigenetic modulators and precisely effect the desired modifications in the genome. The methylases such as DRM1, DNMT2, and MET1 can be fused to dCas9 and lock the target genes in the “off” position. Methylation near promoter regions can lead to little or no transcription and subsequently repress gene expression. Similar effects can be expected while fusing the acetylation- or sumolyation-inducing adaptors. The demethylases such as DME, DML2, and DML3 can also be recruited onto target sites to demethylate any important subsets of enhancers and hence regulate gene expression. Addition of such groups can escalate/downregulate the expression of targeted genes, confirming that the epigenetic marks have profound effects on the transcriptional apparatus. It is possible to observe a synergistic effect when multiple epigenetic modulators are simultaneously linked to dCas9. Molecular Plant 2018 11, 886-898DOI: (10.1016/j.molp.2018.05.008) Copyright © 2018 The Author Terms and Conditions

Figure 5 Combined cis–trans Engineering Approaches for Regulating Gene Expression Patterns. Coordinated TF and promoter engineering requires careful evaluation of the promoter and how it is limited by the binding of specific TFs. Creation of synthetic promoters can be coupled to engineered TFs in a more rational approach whereby the focus can be shifted broadly toward having “inducible” rather than “constitutive” systems. The basic idea is to develop a promoter with a staged output, which is linked to multiple small guide (sg)-RNAs, each having sequence complementarity to a different stress-responsive gene. Here, dCas9 is fused to a transcriptional activator (such as VP16) and is directed to the specific target via the corresponding sgRNA under a particular stress stimulus. Multiple sgRNAs can simultaneously activate multiple genes and significantly elevate the levels of transcription. A unique sgRNA complementary to the promoter region of the dCas9 gene can recruit the dCas9-activator complex to its own promoter and hence autoinduce the gene (dCas9) expression. This multiplex regulation, which is mediated by the sgRNAs-dCas9-activator complex, ensures the control of multiple genes at once without altering the overall gene architecture. Molecular Plant 2018 11, 886-898DOI: (10.1016/j.molp.2018.05.008) Copyright © 2018 The Author Terms and Conditions

Figure 6 A Workflow that Delineates the Methods Adopted to Genetically Reconfigure the Transcriptional Apparatus. While both cis and trans engineering are used in separate approaches to modify the promoter and TFs, respectively, the combined approach can be used for a more pronounced effect. The “CRISPR-Cas9-based bipartite module” can be employed for an efficient, specific, and smarter reframing of the cis–trans configuration in a multiplexed approach. Molecular Plant 2018 11, 886-898DOI: (10.1016/j.molp.2018.05.008) Copyright © 2018 The Author Terms and Conditions